Listeria monocytogenes is a ubiquitous bacterium that can cause serious food-borne infections in pregnant women, newborns and immunocompromised adults. The bacterium grows directly in the cytoplasm of infected host cells and moves rapidly throughout and between infected cells using a form of actin-based motility. The overall goal of this proposal is to understand the mechanism of the actin-based motility of L. monocytogenes. Biochemical and genetic screens will be used to determine whether the ActA protein, a bacterial gene product necessary for virulence and motility, is sufficient to mediate interactions between host cell actin filaments and intracellular bacteria or whether other bacterial proteins are required. Complementary studies will identify host proteins that are necessary for bacterial motility, focusing in particular on host proteins that bind to the bacterial ActA protein. Immunofluorescence and video microscopy techniques will be used to address the question of how the polarity of bacterial movement is established and maintained. Finally, an optical force trap (laser tweezers) will be used to measure the amount of force generated by moving bacteria in infected cells and cell-free extracts in an attempt to determine whether the force necessary for L. monocytogenes movement is provided by a motor protein or by actin polymerization. Successful completion of our research goals would not only give insight into ways of preventing the spread of bacterial parasites such as L. monocytogenes but would also contribute to our understanding of a wide variety of basic biological processes involving actin-based cell movement, including wound healing, immune system responses, and embryonic development. Furthermore, since most tumors are not lethal until the cancer cells move away from the tumor site and invade other tissues, a detailed understanding of the basic mechanisms that regulate actin-based motility may also be important in the development of therapeutic strategies for combating the spread of cancer.